LOCUS       sequence-189822-        5556 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5556)
  TITLE     Bokoch Lab GTPases
REFERENCE   2  (bases 1 to 5556)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pGEX-2T-RhoA-T19N
FEATURES             Location/Qualifiers
     source          1..5556
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     8..25
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        98..175
                     /gene="lacI"
                     /label=lacI promoter
     CDS             176..1258
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(195..214)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1307..1312,1313..1330,1331..1337)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1345..1361
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     1369..1385
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1369..1385
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1381..1908
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(1401..1418)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1401..1417)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1410..1432)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(2010..2015,2016..2031,2032..2038)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2046..2062
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             2085..2738
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2696..2718
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2745..2762
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(2763..2816,2817..2819,2820..3344)
                     /codon_start=1
                     /product="dominant negative mutant of human Rho family 
                     small GTPase RhoA"
                     /label=RhoA (T19N)
                     /translation="MAAIRKKLVIVGDGACGKNCLLIVFSKDQFPEVYVPTVFENYVAD
                     IEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVK
                     HFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKT
                     KDGVREVFEMATRAALQARRGKKKSGCLVL"
     primer_bind     complement(3433..3455)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3600..3618)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3686..3790
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3791..3859,3860..4651)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4009..4028)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4822..5410
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5311..5330
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
       61 tacgcatctg tgcggtattt cacaccgcat aaattccgac accatcgaat ggcgcaaaac
      121 ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa
      181 accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg
      241 cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat
      301 ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt
      361 gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc
      421 gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag
      481 cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct
      541 gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa
      601 tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc
      661 ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat
      721 cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca
      781 taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc
      841 catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat
      901 gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct
      961 gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta
     1021 tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga
     1081 ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc
     1141 actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt
     1201 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     1261 gcaacgcaat taatgtaagt tagctcactc attaggcacc ccaggcttta cactttatgc
     1321 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
     1381 atgaccatga ttacggattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     1441 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     1501 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc
     1561 tttgcctggt ttccggcacc agaagcggtg ccggaaagct ggctggagtg cgatcttcct
     1621 gaggccgata ctgtcgtcgt cccctcaaac tggcagatgc acggttacga tgcgcccatc
     1681 tacaccaacg tgacctatcc cattacggtc aatccgccgt ttgttcccac ggagaatccg
     1741 acgggttgtt actcgctcac atttaatgtt gatgaaagct ggctacagga aggccagacg
     1801 cgaattattt ttgatggcgt tggaattagc ttatcgactg cacggtgcac caatgcttct
     1861 ggcgtcaggc agccatcgga agctgtggta tggctgtgca ggtcgtaaat cactgcataa
     1921 ttcgtgtcgc tcaaggcgca ctcccgttct ggataatgtt ttttgcgccg acatcataac
     1981 ggttctggca aatattctga aatgagctgt tgacaattaa tcatcggctc gtataatgtg
     2041 tggaattgtg agcggataac aatttcacac aggaaacagt attcatgtcc cctatactag
     2101 gttattggaa aattaagggc cttgtgcaac ccactcgact tcttttggaa tatcttgaag
     2161 aaaaatatga agagcatttg tatgagcgcg atgaaggtga taaatggcga aacaaaaagt
     2221 ttgaattggg tttggagttt cccaatcttc cttattatat tgatggtgat gttaaattaa
     2281 cacagtctat ggccatcata cgttatatag ctgacaagca caacatgttg ggtggttgtc
     2341 caaaagagcg tgcagagatt tcaatgcttg aaggagcggt tttggatatt agatacggtg
     2401 tttcgagaat tgcatatagt aaagactttg aaactctcaa agttgatttt cttagcaagc
     2461 tacctgaaat gctgaaaatg ttcgaagatc gtttatgtca taaaacatat ttaaatggtg
     2521 atcatgtaac ccatcctgac ttcatgttgt atgacgctct tgatgttgtt ttatacatgg
     2581 acccaatgtg cctggatgcg ttcccaaaat tagtttgttt taaaaaacgt attgaagcta
     2641 tcccacaaat tgataagtac ttgaaatcca gcaagtatat agcatggcct ttgcagggct
     2701 ggcaagccac gtttggtggt ggcgaccatc ctccaaaatc ggatctggtt ccgcgtggat
     2761 ccatggctgc catccggaag aaactggtga ttgttggtga tggagcctgt ggaaagaact
     2821 gcttgctcat agtcttcagc aaggaccagt tcccagaggt gtatgtgccc acagtgtttg
     2881 agaactatgt ggcagatatc gaggtggatg gaaagcaggt agagttggct ttgtgggaca
     2941 cagctgggca ggaagattat gatcgcctga ggcccctctc ctacccagat accgatgtta
     3001 tactgatgtg tttttccatc gacagccctg atagtttaga aaacatccca gaaaagtgga
     3061 ccccagaagt caagcatttc tgtcccaacg tgcccatcat cctggttggg aataagaagg
     3121 atcttcggaa tgatgagcac acaaggcggg agctagccaa gatgaagcag gagccggtga
     3181 aacctgaaga aggcagagat atggcaaaca ggattggcgc ttttgggtac atggagtgtt
     3241 cagcaaagac caaagatgga gtgagagagg tttttgaaat ggctacgaga gctgctctgc
     3301 aagctagacg tgggaagaaa aaatctggtt gccttgtctt gtgaggatcc ccgaattccc
     3361 gggtcgactc gagcggccgc atcgtgactg actgacgatc tgcctcgcgc gtttcggtga
     3421 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     3481 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     3541 cgcagccatg acccagtcac gtagcgatag cggagtgtat aattcttgaa gacgaaaggg
     3601 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     3661 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3721 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3781 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3841 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3901 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     3961 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     4021 ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     4081 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     4141 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     4201 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     4261 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     4321 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     4381 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     4441 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     4501 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     4561 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     4621 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     4681 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4741 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4801 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4861 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4921 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     4981 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     5041 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     5101 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     5161 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     5221 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     5281 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     5341 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     5401 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     5461 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     5521 tgagtgagct gataccgctc gccgcagccg aacgac
//
